Spray freezing

ABSTRACT

The present invention relates to an improved method for preservation of e.g. microorganisms, especially lactic acid bacteria, said method includes spray freezing.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Stage of InternationalApplication PCT/EP2015/078073, filed Nov. 30, 2015, and claims priorityto Denmark Patent Application No. PA 2014 00697, filed Nov. 28, 2014

FIELD OF INVENTION

The present invention relates to an improved method for drying and/orfreezing proteins or microorganisms, especially lactic acid bacteria,said method includes spraying of a suspension/solution of the protein ormicroorganism into a gas.

BACKGROUND OF INVENTION

Spray drying has previously been used for drying lactic acid bacteria,but without much commercial success. For instance U.S. Pat. No.6,010,725A (Nestle) relates to a process for spray drying microorganismsin a spray drying apparatus having an inlet temperature above 250Degrees Centigrade (° C.). It is stated that at least 10% of themicroorganisms survive the treatment.

Spray freezing has recently been proposed for freezing lactic acidbacteria, but with limited commercial success. Semyonov et al (FoodResearch International 43, 193-202 (2010) have investigated the survivalof Lactobacillus paracasei cells which were microencapsulated by “sprayfreeze drying”, i.e. spray freezing succeeded by freeze drying.Apparently, the bacterial suspension is sprayed directly into nitrogenin its liquid state, which process results in microcapsules having assize distribution between 400 and 1800 micrometers (microns). It isconcluded that bulk freeze drying resulted in slightly higher survivalthan spray freeze drying, and that particles having a size about1000-1400 micrometer result in a higher survival than 400 micrometerparticles.

U.S. Pat. No. 7,007,406 (Wang) discloses a spray-freezing apparatus,where the frozen product is collected on a filter.

All the above spray freezing and drying processes have had limitedcommercial success, especially when the product to be preserved isbacteria cells which should be viable after thawing or rehydrating.

SUMMARY OF INVENTION

The present inventors have surprisingly discovered that bacteria cellscan be preserved very effectively and with a high survival rate by aprocess which includes spray freezing, if:

-   -   the spray freezing step is preceded by a drying step, wherein        the atomized particles are (partly) dried by contacting with a        drying gas;    -   the freezing is carried out by freezing the atomized particles        in a cryogenic gas; and/or    -   the frozen product is collected by means of a cyclone.

Also, the present inventors have observed that the freezing process ofthe invention has unexpected advantages compared to the traditionalfreezing processes, such as:

-   -   improved process economy due to i) less consumption of nitrogen        gas for freezing, compared to pellet freezing, ii) it is        possible to operate a freeze-dryer more efficiently with a        frozen product obtained by the present invention, calculated by        the amount loaded into the freeze dryer, and amount of the final        product obtained after the freeze drying, and iii) a more        compact frozen product is obtained, thereby reducing the need        for freezer space and/or easier transport, and    -   improved removal of liquid if the suspension/solution is        subjected to a pre-drying step before the freezing step;        apparently the evaporated liquid does not form particles in the        freezing step and is removed together with the cryogenic gas,        and/or any formed particles are so small that they are separated        from the desired frozen product, e.g. in a cyclone.

In more details, the present inventors have shown that in conventionalspray freezing of aqueous formulations at ambient atmospheric pressureusing liquid nitrogen and/or very cold nitrogen gas, no drying takesplace during the freezing process at all, thus only offering a smallerparticulate for downstream freeze drying compared to the larger pelletsobtained conventional pelletizing in liquid nitrogen. Conventionally,spray dryers have throughput rates so low that these drying methods donot offer an economical feasible alternative to for example freezedrying. Too high dryer outlet temperatures will destroy and kill mostlactic acid bacteria strains efficiently, as most bacteria living in thehuman gut (such as lactic acid bacteria—hereafter abbreviated LAB,especially anaerobic LAB) will not survive 40° C. for much time.

In an attempt to combine spray drying and downstream freezing of thepartially dehydrated droplets, the present inventors retrofitted aheated (nitrogen) gas distributor around the atomization device placedat the top of a spray freezing chamber. The spray freezing chamber wasmodified so the upper half would sustain temperatures well above thefreezing point of most aqueous LAB suspensions and in the lower half ofthe combined spray drying/freezing chamber, liquid nitrogen was injectedin order to accomplish spray freezing of the partially dehydrateddroplets generated by the atomization device in the top of the chamber.

After freezing, the microscopic ice crystals and the much larger frozenproduct pellets was pneumatically transported to a downstream cyclone bythe combined amounts of nitrogen gas from both the spray drying anddownstream spray freezing sections of the spray chamber. The microscopicice crystals was so small the downstream cyclone separator was not beable to separate them completely from the nitrogen gas, whereas the muchlarger frozen product pellets was efficiently be separated from thenitrogen gas as expected. Thus, it has surprisingly turned out that itis advantageous to use a cyclone for separation of the frozen particlesfrom the cryogenic gas. The present inventors discovered that any watervapour from the initial spray drying process will shock freeze intomicroscopic ice crystals along with freezing of the partially dehydrateddroplets arriving from the upper warm half of the spray chamber almostinstantly upon contact with the liquid nitrogen spray in the lower halfof the spray chamber. The recovered partially dehydrated/frozen productpellets (in various sizes in the range 50 to 400 microns) typicallydisplayed a decrease in moisture content in the range of 0.5-50% (w/w),depending on drying gas rate/temperature and product feed rate and wasfound to have approx. 15% higher bulk density compared to the muchlarger commercially available frozen product pellets (3000-10000 micronsor 3-10 mm), generated by conventional liquid nitrogen pelletizing, thusincreasing the overall capacity of any freeze dryer unit used for thedownstream final drying step of the product.

The inventors continued their investigations, and performed downstreamfinal freeze drying of the recovered partially dehydrated/frozen productpellets and found the new invention which combines spray drying/freezingprocess yields surprisingly high bacterial survival and activity afterdrying.

To their surprise, they also found the freeze dried micropellets shrinksubstantially compared to freeze dried larger frozen product pellets,generated by conventional liquid nitrogen pelletizing, in fact the finalbulk density of the freeze dried product was about 2½ times higher thanconventional freeze dried powders. The density increase translates intomuch lower product porosity and thus improved product stability and manyother advantages in the final application of the product.

The present inventors discovered that the best result was obtained whenthe drying gas used in spray dryer/freezer was free of oxygen, and wetherefore contemplate that the gas should preferably be an inert gaslike Nitrogen or any noble gas like Helium, Argon and Neon etc. The bestresult is presently obtained by combining the use of an inertdrying/cryogenic/conveying gas with drying pressures at ambientpressure, but it is contemplated that it will be an advantage to carryout the process at pressures below or above ambient pressure.

The spray drying/freezing method of the invention results in improvedsurvival and stability of the LAB, and combined with the dryness of theproduced LAB containing powders this yields an economical feasiblepre-drying/freezing process for heat- and oxygen labile LAB containingproducts.

Further, it has turned out that the product of the combineddrying/freezing process followed by conventional freeze drying, i.e. thedried powder, has several unexpected advantages relative to freeze driedmuch larger frozen product pellets, generated by conventional liquidnitrogen pelletizing, containing the same heat-labile material, e.g.improved survival (more active material, i.e. higher yield), easierapplicability (the powder is easier to disperse in an aqueous solutionor suspension, such as milk).

The invention does not limit itself for LAB drying alone: Most livebacterial/viral strains, large macro-molecules like proteins/peptidesand other biopharmaceutical/biological products in general will benefitfrom this oxygen-free and low temperature pre-drying/freezing method.Thus, the present invention comprises these embodiments.

In accordance with the above surprising findings, the present inventionin a first aspect relates to a process for preserving heat labilematerial such as proteins or microorganisms by freezing and optionallydrying a solution or suspension containing the material, characterizedby contacting droplets of the suspension or solution with a drying gasand subsequent contacting the (partially) dried droplets with acryogenic gas.

In a second aspect, the present invention relates to a productobtainable by the process of the first aspect.

In a third aspect, the present invention relates to an apparatus usablein the process of the first aspect, such as an apparatus comprising achamber with i) an atomizing means for atomizing the suspension, ii) aninlet for a drying gas (optionally integrated in the atomizing means),iii) an inlet for a cryogenic gas, and iv) an outlet optionallyconnected with a cyclone, e.g. an apparatus substantially as depicted onFIG. 1 or 4.

DETAILED DISCLOSURE

In a first aspect, the present invention relates to a process forpreserving (freezing and/or drying) microorganisms (esp. LAB (LacticAcid Bacteria)) or proteins, said process involves subjecting droplets(e.g. an atomized suspension) containing the microorganism/protein to acryogenic gas. Interesting embodiments of this first aspect are:

1: A process for preserving microorganisms (esp. LAB (Lactic AcidBacteria)), such as preserving by freezing and optionally drying,comprising the following steps:

-   -   a) Preparing droplets (preferably having a size from 10 to 500        micrometer, such as a size in the ranges: 15 to 400, 20 to 350,        50 to 350, 100 to 350, 20 to 300, 20 to 200, 50 to 300, 50 to        200, 100 to 300, or 100 to 200, measured as the Dv90 value (Dv90        is defined as the maximum particle diameter below which 90% of        sample volume exists, according to the ISO 13320:2009 standard        for Particle size analysis—Laser diffraction methods, measured        in micrometer)) of an aqueous (or liquid) suspension containing        the microorganisms (preferably having a content of at least        1.0E+8 microorganisms per ml), e.g. by spraying (atomizing) the        suspension; and    -   b) Optionally contacting the droplets with a drying gas        (preferably having a temperature in the range from 20° C. to        250° C., and/or preferably for a time period of from 1 second to        120 seconds); and    -   c) Contacting the droplets, such as the droplets resulting from        the preceding step, with a cryogenic gas (preferably having an        temperature in the range from −20 to −150° C. or from −50 to        −100° C., and/or preferably for a time period of from 1 sec to        120 sec); and    -   d) optionally subjecting the resulting frozen product from the        preceding step to a further drying step, such as drying under        reduced pressure, e.g. freeze-drying; and    -   e) optionally packaging the microorganisms, such as in an        air-tight and/or moisture-tight package (optionally together        with microorganisms of a different strain).        2: A process for drying a microorganism (esp. a LAB) containing        suspension, characterized in that:        i) an aqueous suspension containing microorganisms (preferably        having a concentration of at least 1.0E+8 microorganisms per ml)        is sprayed into a cryogenic gas (preferably having a temperature        in the range from −20 to −150° C.) in a spray chamber; and        ii) the frozen product from step a) is collected and freeze        dried until a water activity of less than 0.20 is achieved.        3: A process for drying a microorganism (esp. a LAB) containing        suspension, characterized in that:        i) an aqueous suspension containing microorganisms (preferably        having a concentration of at least 1.0E+8 microorganisms per ml)        is sprayed into a drying gas (preferably having a temperature in        the range from 30 to 200° C.) in a spray chamber;        ii) the product from step al) is contacted with a cryogenic gas        (preferably having a temperature in the range from −20 to −150°        C.) in a chamber; and        iii) the frozen powder from step a) is collected and freeze        dried until a water activity of less than 0.20 is achieved.        4: A process for drying (or removing liquid from) a solution or        suspension containing proteins (such as enzymes, hormones, human        proteins, therapeutically active proteins or lipoproteins) or        microorganisms (such as bacteria, LAB, yeasts, fungi, plant        cells, animal cells, or vira), characterized by:    -   a) Preparing droplets (preferably having a size from 10 to 500        micrometer, such as a size in the ranges: 15 to 400, 20 to 350,        50 to 350, 100 to 350, 20 to 300, 20 to 200, 50 to 300, 50 to        200, 100 to 300, or 100 to 200, measured as Dv90 in microns) of        the suspension or solution, e.g. by spraying the solution or        suspension; and    -   b) Optionally contacting the droplets with a drying gas        (preferably having a temperature in the range from 20 degrees C.        to 250 degrees C., and/or preferably for a time period of from 1        second to 120 seconds); and    -   c) Contacting the droplets obtained in step a) or b) with a        cryogenic gas (preferably having an temperature in the range        from −20 to −150 degrees C. or from −50 to −100, and/or        preferably for a time period of from 1 sec to 120 sec); and    -   d) subjecting the resulting frozen product from the preceding        step to a further drying step, such as drying under reduced        pressure, e.g. freeze-drying; and    -   e) Optionally packaging the product, such as in an air-tight        and/or moisture-tight package.        5: A process for freezing a suspension containing microorganisms        (esp. LAB), by:    -   Spraying the suspension into a chamber containing a drying gas        (preferably having a temperature in the range from 20 degrees C.        to 250 degrees C., and/or preferably so the suspension is in        contact with the gas for a time period of from 1 second to 120        seconds); and    -   Freezing the product from the preceding step by contacting with        a cryogenic gas in a (preferably having an temperature in the        range from −20 to −150 degrees C. or from −50 to −100, and/or        preferably so the product is in contact with the cryogenic gas        for a time period of from 1 sec to 120 sec); and    -   optionally packaging the frozen suspension, such as in an        air-tight and/or moisture-tight package.

It the above processes, it should be understood that the drying step isperformed for a time sufficient for achieving the desired degree ofdrying, and the freezing is performed for a time sufficient for acomplete freezing can be achieved, i.e. the product should be completelyfrozen. The skilled person knows how to obtain the suitable conditionsin e.g. a two-chamber (two-zone) spray tower, and he knows how tocalculate the height of the spray tower so the sprayed suspension has asuitable passage time through the drying chamber/zone and freezingchamber/zone, resp.

In the above processes, the droplets are preferably prepared byspraying. The spraying may be carried out by means of a spray nozzle(atomizing device), such as an ultrasound nozzle, a pressure nozzle, atwo-fluid nozzle (e.g. using N2 as atomizing gas), or a rotatingatomizing device, the atomizing preferably resulting in droplets havinga size from 10 to 500 micrometer, such as having a size selected fromone of the following ranges: 15 to 400, 20 to 350, 50 to 350, 100 to350, 20 to 300, 20 to 200, 50 to 300, 50 to 200, 100 to 300, or 100 to200, measured as Dv90 values in micrometer.

The frozen product (e.g. powder) may be collected by means of a cyclone,or an electrostatic filter. A cyclone is presently preferred, such as acyclone operated with a with a maximum differential pressure drop acrossthe cyclone of 10 mm to 300 mm water column, or 50 to 200 mm watercolumn, or approx. 100 mm water column.

It is presently preferred that the spray drying and/or that the freezingstep takes place under a pressure between 60 and 200 kPa, such asbetween 80 and 150 kPa or between 60 and 120 kPa, between 70 and 110kPa, or between 105 and 140 kPa.

Advantageously, the final drying step (of the frozen product) may takeplace under reduced pressure, such as by freeze-drying, preferably to anaw below 0.20.

Other interesting embodiments of the processes of the first aspect are:

-   -   A process, wherein the “retention time” is less than 2 minutes        in the spray dryer, and preferably the resulting spray-dried (or        partly dried) product is directly introduced into a freezing        chamber, such as by using an apparatus wherein the dried (or        partially dried) product from the spray drying is transferred        into the freezing chamber by means of the gravity.    -   A process, wherein the spray-drying step is carried out at with        a drying gas inlet temperature of at most 300° C.,        advantageously between 20° C. and 250° C. and more preferably        between 100° C. and 200° C.    -   A process, wherein the spray-drying step takes place at a        temperature in the range from 20° C. to 200° C., and/or        preferably for a time period of from 1 second to 120 seconds).    -   A process, wherein after the spray-drying step, the droplet has        a size of between 20 and 400 microns and preferably between 50        and 300 microns.    -   A process, wherein after the spray-drying step the water content        of the droplet is at least 20 percent by weight reduced compared        to the starting suspension, preferably at least 40%, at least        60% or at least 75%.    -   A process, wherein after the drying step the liquid (e.g. water)        content of the droplet is at least 5% by weight reduced compared        to the starting suspension/solution, preferably at least 10%.    -   A process, wherein after the drying step the liquid (e.g. water)        content of the droplet is between 20% and 75% by weight, with        respect to the total weight of the droplet.    -   A process, wherein after the spray-drying step the water content        of the droplet is between 20% and 85% by weight, (preferably        between 30% and 80%, or between 40% and 75% percent by weight),        with respect to the total weight of the droplet.    -   A process, wherein the duration of the freeze-drying step is a        time suitable for obtaining a powder whose residual water        content of the powder is at least 0.15.    -   A process, wherein the drying gas contains less than 5% oxygen,        such as less than 2%.    -   A process, wherein the cryogenic gas contains less than 5%        oxygen, such as less than 2%.    -   A process, wherein the drying gas is selected from the group        consisting of an inert gas (such as Nitrogen), a noble gas (such        as Helium, Argon or Neon) etc., carbon dioxide, and an alkane        gas (such methane), and a mixture thereof.    -   A process, wherein the cryogenic gas is selected from the group        consisting of an inert gas (such as Nitrogen), a noble gas (such        as Helium, Argon or Neon) etc., carbon dioxide, and an alkane        gas (such methane), and a mixture thereof.    -   A process, wherein the cryogenic gas has (an inlet) temperature        in the range from −50 to −250° C. or in the range from −80 to        −160° C.    -   A process, wherein the solution or suspension further comprises        an additive that stabilizes the material/microorganism. The        additive may be selected from the group consisting of: Inositol,        lactose, sucrose, trehalose, inulin, maltodextrin, skimmed milk        powder, yeast extract, casein peptone, inosine,        inosinemonophospate, monosodium glutaminate, sodium caseinate,        and sodium ascorbate, polysorbate. It is presently preferred        that the ratio heat labile material (proteins or        microorganisms):additive is within the range from 1:0.5 to 1:5,        such as from 1:1 to 1:4 or from 1:1½ to 1:3, (w/w of the dry        weights).    -   A process, wherein the microorganism is selected from the group        consisting of: a yeast (eg Saccharomyces), a lactic acid        bacterium, a Streptococcus species, a Lactobacillus species, a        Lactococcus species, a Leuconostoc species, a Bifidobacterium        species, an Oenococcus species, a Bacillus species.    -   A process, wherein the microorganism is selected from the group        consisting of Streptococci species, such as Streptococcus        thermophilus.    -   A process, wherein the microorganism is selected from the group        consisting of Bifidobacterium species, such as B. animalis ssp.        lactis or B. longum.    -   A process, wherein the microorganism is selected from the group        consisting of Lactobacillus species, such L. acidophilus or L.        bulgaricus.    -   A process, wherein the microorganism is selected from the group        consisting of Lactococcus species, such as L. lactis or L.        cremoris    -   A process, wherein the microorganism is selected from the group        consisting of Bacillus species, such as B. subtilis.    -   A process, which process takes place in an apparatus according        to the invention, such as in an apparatus substantially as        depicted in the FIGS. 1, 4, 5, and/or 6.

In a second aspect, the present invention relates to a productobtainable by any of the above processes. In a presently preferredembodiment, the product may be packaged (e.g. in an airtight container).

In a specific embodiment, an additive is added to the heat labilematerial before spraying, especially if the material is to be subjectedto freeze drying. The additive is preferable a mixture of differentcompounds that protect the material during freezing. A preferredadditive comprises 5-50% ascorbic acid (or ascorbate), 5-50% inositol,and 5-50% glutamate (in dry form, w/w). Such an additive is also a partof the present invention.

In a third aspect, the present invention relates to an apparatus usablein any of the above processes, such as an apparatus comprising a chamberwith i) an atomizing means for atomizing the suspension, ii) an inletfor a drying gas (optionally integrated in the atomizing means), iii) aninlet for a cryogenic gas, and iv) an outlet optionally connected with acyclone, e.g. an apparatus substantially as depicted on FIG. 1 or FIG.4.

An interesting embodiment of the third aspect is an apparatus of theinvention, which comprises a two-chamber tower (wherein the firstchamber is placed over the second chamber) wherein the first (upper)chamber (11) comprises i) an atomizing means for atomizing thesuspension (5), and ii) an inlet for the drying gas (optionallyintegrated in the atomizing means); and the second (lower) chamber (12)comprises i) an inlet for a cryogenic gas and ii) an outlet coupled to afirst cyclone (14). It should be understood that by placing the firstchamber over the second chamber, the chambers are linked so that the(partially) dried particles will by means of the gravity drop from thefirst chamber into the second chamber.

Yet an embodiment relates to an apparatus (such as a spray tower)comprising an first (upper) chamber (11) and a second (lower) chamber(12), wherein the upper chamber comprises

-   -   means for atomizing a suspension or solution (5); and    -   an inlet for a drying gas (1), and    -   means (3) for heating the drying gas to a temperature in the        range 20° C. to 250° C.; and wherein the lower chamber comprises    -   an inlet for a cryogenic gas (4) (adapted for a gas having a        temperature in the range −50 to −250° C.) and optionally a tank        for storing the cryogenic gas; and    -   an outlet for the frozen product, said outlet being connected to        a cyclone (13); and wherein the upper chamber is placed so the        (partly) dried particles drop into the lower chamber for        subsequent freezing.

An interesting embodiment is an apparatus, such as an apparatusaccording to the preceding embodiment, comprising a first (upper)chamber and a second (lower) chamber, wherein the upper chambercomprises

-   -   means for atomizing a suspension or solution; and    -   means of injection of drying gas; and    -   means for supplying the drying gas at a temperature in the range        20° C. to 250° C.;        And wherein the lower chamber comprises    -   means for injection of a cryogenic gas; and    -   means for supplying the cryogenic gas at a temperature in the        range −50 to −250° C.; and    -   an outlet for the frozen product, said outlet preferably being        connected to a cyclone.

In a presently preferred embodiment, first chamber is connected to aheater for heating the drying gas. It is also preferred that the secondchamber is connected to a tank adapted to a cryogenic gas.

In case the apparatus should be operated at a pressure different fromambient pressure, the apparatus should comprises means for lowering thepressure (e.g. to a pressure below 0.9 bar) in the first chamber and/orin the second chamber, or means for increasing the pressure (e.g. to apressure above 1.1 bar) in the first chamber and/or in the secondchamber.

It is presently preferred that the first chamber has a height thatallows at least 5% of the liquid in the suspension/solution to evaporateduring the passage, and wherein the second chamber has a height thatallows a complete freezing of the product entering from the upperchamber. In an embodiment, the first chamber is essential cylindricaland has a diameter of 0.5 to 5 m and a height of 1 to 4 times thediameter. In yet an embodiment, the second chamber is essentialcylindrical and has a diameter of 0.5 to 5 m and a height of 1 to 2times the diameter. In an preferred embodiment, the first and secondchamber is connected so the first chamber is the upper part of and thesecond chamber is the lower part of an essential cylindrical structurewhich and has a diameter of 0.5 to 5 m and a total height of 2 to 6times the diameter.

The apparatus may further comprise a second cyclone coupled to thematerial outlet of the first cyclone. Advantageously, the apparatus ofthe invention further comprises a gas inlet between the first cycloneand the second cyclone, so that the gas will convey the materialdischarged from the first cyclone to the second cyclone. The gas may bea cryogenic gas, and/or a gas which contains less than 5% oxygen, suchas less than 2%. The gas may be selected from the group consisting of aninert gas (such as Nitrogen), a noble gas (such as Helium, Argon orNeon) etc., carbon dioxide, and an alkane gas (such methane), and amixture thereof.

In a preferred embodiment, the following apparatus is used as described,cf. FIG. 1:

A primary inert gas supply (a) is connected to the inlet of a gas heater(b). The gas heater is connected to the combined spray drying/freezingchamber top inlet (d) and heats the inert gas to an the inlettemperature set by the inlet control loop (c).

The liquid feed supply (e) is connected to the suction side of a liquidfeed pump (f) which pumps the liquid formulation to the atomizationdevice (g) on the top of the combined spray drying/freezing chamber (d).The atomization device (g) sprays the liquid feed into a cloud ofaerosol droplets which dries into partially dehydrates droplets byconsuming the heat supplied by the heated inert gas.

As the partially dehydrated droplets leaves the upper warm section ofthe combined spray drying/freezing chamber (d) together with the nowcooled and moist inert gas, both rapidly cools down as they enter theliquid nitrogen cooled lower section of the combined spraydrying/freezing chamber (d). Cold nitrogen gas pulls microscopic watercrystals and now frozen partially dehydrated product towards the chamberoutlet and pneumatically transports the particulates towards adownstream cyclone separator (h) where the frozen partially dehydratedparticles are separated from the inert gas and microscopic ice crystals.The cold and inert gas and microscopic ice crystals are led to a warmwater scrubber unit (i) and downstream an exhaust fan (j) creates therequired chamber pressure set by the chamber pressure control loop (k).To improve the control of the combined drying/freeze chamber outlettemperature an outlet temperature control loop (l) is used to controlthe liquid nitrogen injection (y).

In a last aspect, the invention relates to the use of the apparatus ofthe invention, wherein a drying gas (having a temperature in the range20° C. to 250° C.) and a liquid containing a protein or a microorganismis sprayed into the upper chamber; and a cryogenic gas (having atemperature in the range −50 to −250° C.) is sprayed into the lowerchamber.

Definitions

As used herein, the term “lactic acid bacterium” (LAB) designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of the strict anaerobic bacteria, bifidobacteria,i.e. Bifidobacterium spp., are generally included in the group of lacticacid bacteria. These are frequently used as food cultures alone or incombination with other lactic acid bacteria. Interesting species of LABare selected from the group comprising the strains of the species andsubspecies Bifidobacterium bifidum, Bifidobacterium lactis,Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalisssp. lactis, Lactobacillus reuteri, Lactobacillus acidophilus,Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbruckiibulgaricus, Lactobacillus rhamnosus, Streptococcus thermophilus,Streptococcus salivarius, Lactococcus lactis, Lactobacillus pentoceus,Lactobacillus buchneri, Lactobacillus brevis, Pediococcus pentosaceus,Pediococcus acidilactici, Pediococcus pervulus, Propionibacteriumfreudenreichi, Propionibacterium jenseni and mixtures thereof.

Also the term LAB includes the strains Lactobacillus rhamnosus GG (LGG),Lactobacillus casei (LC-431), Lactococcus lactis (R704), Bifidobacteriumanimalis ssp. Lactis (BB-12), Streptococcus thermophilus (ST-Fe 2),Lactobacillus bulgaricus (LB CH-2)

In the present context, the term “spray drying” means partially removingliquid (e.g. water) from a solution or suspension, i.e. a concentrationof the desired microorganism or protein containing solution/suspension.In the spray drying process of the invention, it is presently preferredthat 10% to 70% of the water in the droplet is removed, and/or the ratioof dry heat labile material in the product (microorganism/protein) afterspray drying has increased more than 25% but less than 400% (compared tothe ratio of the starting material). Thus, the product after the spraydrying is preferably a liquid or a wet product, and not a dry powder.Presently preferred is a liquid (e.g. aqueous) suspension with amicroorganism, or a liquid (e.g. aqueous) solution with a protein. Bynot drying the product completely, less heat labile material isinactivated. The skilled person knows how to secure that the material isnot inactivated, e.g. by lowering the temperature of the drying gas,and/or reducing the contact time with the drying gas, and/or by reducingthe distance the droplets have to travel in the spray drying chamber.

If the product after spray freezing is subjected to freeze drying, it ispresently preferred that the water activity (aw) of the resultingproduct is below 0.2.

In the present context, the term “packaging” (a suitable amount of) thefrozen or dried microorganism in a suitable packaging relates to thefinal packaging to obtain a product that can be shipped to a customer. Asuitable packaging may thus be a container, bottle or similar, and asuitable amount may be e.g. 1 g to 30000 g, but it is presentlypreferred that the amount of the microorganism in a package is from 50 gto 10000 g.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

FIGURES

FIG. 1 depicts a preferred spray drying equipment that can be usedaccording to the invention.

(a) drying gas supply (b) drying gas heater (c) Inlet temperaturecontrol loop (d) Combined spray drying/freezing chamber (e) Liquid feedsupply (f) Liquid feed pump (g) Atomization device (h) Cyclone separator(i) Warm water scrubber unit (j) Exhaust fan (k) Chamber pressurecontrol loop (l) Outlet temperature control loop (m) powder discharge(y) cryogenic gas inlet

FIG. 2 depicts the stability data for the strain ST-44 after 3 monthsstorage at −20° C. and at +5° C., cf. example 4.

FIG. 3 depicts the stability data for the strain ST-4895 after 3 monthsstorage at −20° C. and at +5° C., cf. example 2.

FIG. 4 depicts a spray drying/freezing apparatus according to theinvention

1. Drying gas supply 2. Supply fan 3. Heater 4. Cryogenic gas supply 5.Nozzle (with optionally gas supply shown) 6. Liquid feed 7. Liquid feedtank 8. Protein or Microorganism suspension, optional withcryoprotectant 9. Water inlet 10. Liquid feed pump 11. Drying chamber12. Freezing chamber 13. Frozen powder discharge 14. Cyclone 15. Exhaustfan 16. Exhaust gas T. Temperature regulator P. Pressure regulator F.Drying gas regulator

FIGS. 5 and 6 depicts preferred embodiments of the apparatus

EXPERIMENTAL Example 1

A sample of 1281 g of Streptococcus thermophilus (strain ST-Fe 2)concentrate was kept at <5° C. This contained 1.7E+11 CFU/g with approx.12.8% (w/w) dry solids. Parallel to this 579 g of solution was preparedby adding the following ingredients to 470 g of cold tap water (approx.10° C.) under agitation: 33 g sodium ascorbate, 32 g sodium caseinate,22 g inositol and 22 g monosodium glutamate (MSG).

The sample and the additive solution were mixed. This resulted in 1.86kg of liquid formulation with approx. 14.6% (w/w) dry solids to be spraydried. This liquid formulation contained now approx. 1.2E+11 CFU/g andwas kept cold (<5° C.) throughout the test.

A GEA Niro Mobile Minor laboratory spray dryer was modified toaccommodate spray drying using two 380 mm top extension sectionsfollowed by liquid nitrogen injection in the lower fixed section of thestandard spray chamber to accommodate in-situ freezing of the partiallydehydrated product droplets arriving from the upper section of thechamber. The upper spray drying section was supplied with heated purenitrogen drying gas and the lower freezing section was supplied withliquid nitrogen capable of generating a frozen particulate colder than−100° C.

The upper spray dryer section inlet temperature was kept at 190° C.,using a nitrogen drying gas kept at a mass flow-rate of approx. 100kg/h. A 2-fluid nozzle (Schlick 0-2) was used for the atomization of theabove mentioned liquid formulation, using an atomization gas flow ofapprox. 5 kg/h (Nitrogen) equivalent to an atomization pressure of 0.8Bar(g)

The liquid formulation was sprayed into the upper spray dryer section.The feed-rate was kept at 2 kg/h and the spray drying/freezing chamberoutlet temperature was kept in the range −140 to −110° C.

A free-flowing frozen powder with an average particle size of 105 micronwas collected below the downstream cyclone. After 55 min. about 1100 gof partially dehydrated frozen formulation has been collected, whichcorresponds to a yield of about 70%. The moisture content was now 18.5%(w/w) measured as total volatiles on a Sartorious IR at 115° C. Thiscorresponds to a reduction of the total water amount in our product ofapprox. 24% (w/w).

The obtained partially dehydrated frozen powder contained now 1.5E+11CFU/g. The frozen powder was freeze dried, performed at a chamberpressure of 0.3 mbar with temperature increasing from −42° C. to 32° C.with 1.5° C./min. The freeze drying was ended when the weight of theproduct has been constant/stable for at least 2 hours. The dried producthad an acceptable stability after 3 months storage at 5° C. (pH 5.6 asmeasured using standard CINAC analysis).

Example 2

Example 1 was repeated using the same equipment, conditions and additivesolution, but with the strain ST-4895. Thus a sample of 1281 gStreptococcus thermophilus strain ST-4895 concentrate was mixed with 579g of additive solution, resulting in 1.86 kg of liquid formulation withapprox. 14.6% (w/w) dry solids to be spray dried. This liquidformulation contained now approx. 1.2E+11 CFU/g. After drying andfreezing, a frozen powder was obtained.

The spray-frozen powder was freeze dried (cf. example 1). The stabilityof the dried product was compared with a freeze-dried product obtainedfrom a “standard” pellet-frozen concentrate of ST-4895. Performance ofthe freeze dried products was examined by using standard CINAC analysis.For three months stability data, see FIG. 3. The product produced by theprocess according to present invention is stable, even if compared withthe pellet-frozen product.

Example 3

Example 1 was repeated using the same equipment, conditions and additivesolution, but with the Streptococcus thermophilus strain ST-143. Thus, asample of 1281 g of Streptococcus thermophilus (strain ST-143)concentrate was mixed with 579 g of additive solution. This resulted in1.86 kg of liquid formulation with approx. 14.6% (w/w) dry solids to bespray dried. This liquid formulation contained now approx. 1.2E+11CFU/g. After drying and freezing, a frozen powder was obtained.

Example 4

Example 1 was repeated using the same equipment, conditions and additivesolution, but with the Streptococcus thermophilus strain ST-44. Thus, asample of 1281 g of strain ST-44 concentrate was mixed with 579 g ofadditive solution.

This resulted in 1.86 kg of liquid formulation with approx. 14.6% (w/w)dry solids to be spray dried. This liquid formulation contained nowapprox. 1.2E+11 CFU/g. After drying and freezing, a frozen powder wasobtained.

The spray-frozen powder was freeze dried as in example 1, and thestability of the dried product was compared with a product obtained byfreeze drying a pellet-frozen concentrate of ST-44 (method as in example2). For three months stability data, see FIG. 2. The product producedaccording to the present invention is stable, even if compared to thepellet-frozen product.

Example 5

A sample of 2640 g of Bifidobacterium animalis ssp. lactis (strainBB-12®) concentrate was kept at <5° C. This contained 2E+11 CFU/g withapprox. 14.5% (w/w) dry solids. Parallel to this 1080 g of solution wasprepared by adding the following ingredients to 876 g of cold tap water(approx. 10° C.) under agitation: 60 g sodium ascorbate, 79 g skimmedmilk powder, 33 g inositol and 33 g MSG. The sample and the additivesolution were mixed. This resulted in 3.72 kg of liquid formulation withapprox. 15.7% (w/w) dry solids to be spray dried. This liquidformulation contained now approx. 1.4E+11 CFU/g and was kept cold (<5°C.) throughout the test. After drying and freezing preformed as inexample 1, a frozen powder was obtained. The frozen powder was freezedried, and the dried product had an acceptable cell count after 3 monthsstorage at 5 C (2.9E+11 CFU/g).

Example 6

A sample of 1145 g of Lactobacillus bulgaricus (strain LB CH-2)concentrate was kept at <5° C. This contained 1.1E+11 CFU/g with approx.11.5% (w/w) dry solids. Parallel to this 375 g of solution was preparedby adding the following ingredients to 282 g of cold tap water (approx.10° C.) under agitation: 27 g sodium ascorbate, 36 g skimmed milkpowder, 15 g inositol and 15 g MSG. The sample and the additive solutionwere mixed. This resulted in 1.52 kg of liquid formulation with approx.14.7% (w/w) dry solids to be spray dried. This liquid formulationcontained now approx. 8.5E+10 CFU/g and was kept cold (<5° C.)throughout the test. After drying and freezing preformed as in example1, a frozen powder was obtained. The frozen powder was freeze dried, andthe dried product had an acceptable stability after 3 months storage at5 C (pH 6 as measured using standard CINAC analysis).

REFERENCES

-   EP1234019B1 (Danisco A/S)-   U.S. Pat. No. 6,010,725A (Nestle SA)-   Semyonov et al (Food Research International 43, 193-202 (2010)-   U.S. Pat. No. 7,007,406 (Wang)-   WO15063090A2, WO14029758A1, WO14029783A1 (Chr Hansen A/S)-   ISO 13320:2009 standard for Particle size analysis—Laser diffraction    methods

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

The invention claimed is:
 1. A process for removing liquid from asolution or suspension containing microorganisms, comprising: (a)preparing droplets of a solution or suspension containing microorganismsby spraying the solution or suspension; (b) spray-drying the droplets bycontacting the droplets with a drying gas; (c) freezing the dropletsobtained in step (b) with a cryogenic gas to obtain a frozen product;and (d) freeze-drying the frozen product under reduced pressure to awater activity (a_(w)) below 0.20, to produce a freeze-dried product. 2.The process of claim 1, wherein the microorganisms comprise lactic acidbacteria (LAB).
 3. The process of claim 2, wherein: spray-drying step(b) comprises spraying an aqueous suspension containing the LAB into adrying gas in a spray chamber; and freezing step (c) comprisescontacting the product resulting from step (b) with a cryogenic gas in achamber to obtain a frozen powder as the frozen product.
 4. The processof claim 2, wherein: spray-drying step (b) comprises spraying a liquidsuspension containing the LAB into a chamber containing a drying gas;and freezing step (c) comprises freezing the droplets resulting fromstep (b) by contacting the droplets with a cryogenic gas in a chamber toobtain a frozen suspension as the frozen product.
 5. The process ofclaim 1, wherein the spraying of step (a) is carried out by passing thesolution or suspension through a spray nozzle or a rotating atomizingdevice, wherein the spray nozzle or rotating atomizing device results indroplets having a size of from 10 to 500 micrometers, measured as Dv90values in micrometers.
 6. The process of claim 1, wherein the frozenproduct is collected by a cyclone having a maximum differential pressuredrop across the cyclone of about 100 mm water column, or anelectrostatic filter.
 7. The process of claim 1, wherein thespray-drying step (b) and freezing step (c) are independently conductedat a pressure in the range of from 60 to 200 kPa.
 8. The process ofclaim 1, wherein spray-drying step (b) is conducted with a retentiontime of less than 2 minutes in a spray dryer, and wherein the resultingproduct is directly introduced into a freezing chamber.
 9. The processof claim 1, wherein spray-drying step (b) is carried out with a dryinggas inlet temperature of at most 300° C.
 10. The process of claim 1,wherein spray-drying step (b) is conducted at a temperature in the rangefrom 20° C. to 250° C.
 11. The process of claim 1, wherein, afterspray-drying step (b), the droplets have a size of between 20 and 400microns measured as Dv90 values.
 12. The process of claim 1, wherein,after spray-drying step (b), the liquid content of the droplets isreduced by at least 5% by weight as compared to the liquid content ofthe starting suspension or solution.
 13. The process of claim 12,wherein, after spray-drying step (b), the liquid content of the dropletsis between 20% and 85% by weight of the total weight of the droplets.14. The process of claim 1, wherein the drying gas and the cryogenic gaseach independently contain less than 5% oxygen.
 15. The process of claim1, wherein the drying gas and the cryogenic gas are independentlyselected from the group consisting of an inert gas, a noble gas, carbondioxide, an alkane gas, and mixtures of two or more thereof.
 16. Theprocess of claim 1, wherein the cryogenic gas has an inlet temperaturein the range of from −50 to −250° C., and/or the cryogenic gas has atemperature of between −20° C. and −150° C. during the freezing step.17. The process of claim 1, wherein the solution or suspension furthercomprises an additive selected from inositol, lactose, sucrose,trehalose, inulin, maltodextrin, skimmed milk powder, yeast extract,casein peptone, inosine, inosinemonophospate, glutamine and saltsthereof, casein and salts thereof, ascorbic acid and salts thereof, andpolysorbate.
 18. The process of claim 17, wherein the ratio ofmicroorganisms to additive is from 1:0.1 to 1:10 (w/w of the dryweights).
 19. The process of claim 1, wherein the microorganisms areselected from a yeast, a Streptococcus species, a Lactobacillus species,a Lactococcus species, a Leuconostoc species, a Bifidobacterium species,an Oenococcus species, and a Bacillus species.
 20. The process of claim1, wherein the process is carried out in an apparatus comprising atwo-chamber tower, the tower comprising: a first (upper) chambercomprising (i) an atomizer adapted to atomize the suspension orsolution, and (ii) an inlet for the drying gas; and a second (lower)chamber comprising (iii) an inlet for the cryogenic gas, and (iv) anoutlet coupled to a cyclone; wherein the drying gas having a temperaturein the range from 20° C. to 250° C. and the suspension or solution aresprayed into the first (upper) chamber, and the cryogenic gas having atemperature in the range of from −50 to −250° C. is sprayed into thesecond (lower) chamber.
 21. The process of claim 1, wherein the processis carried out in an apparatus comprising a chamber having (i) anatomizer for atomizing the solution or suspension, (ii) an inlet for thedrying gas, (iii) an inlet for the cryogenic gas, and (iv) an outlet.22. The process of claim 1, further comprising packaging thefreeze-dried product.
 23. The process of claim 21, wherein the inlet forthe drying gas is integrated in the atomizer.
 24. The process of claim21, wherein the outlet is connected to a cyclone.
 25. A product obtainedby the process of claim
 1. 26. The product of claim 25, packaged in anairtight container.